A chemical method of sequencing DNA molecules is disclosed in Maxam, Allan M. and Walter Gilbert, "A New Method for Sequencing DNA," Proc. Natl. Acad. Sci. USA, Vol. 74, No. 2, pp. 560-564, February, 1977, the entire contents of which are incorporated herein by reference. The Maxam/Gilbert method provides for the terminal labeling of several identical DNA strands with radioactive tracers and then breaking the strands at each base into fragments using chemical agents. The relative lengths of the labeled fragments identify the position of the associated base in the strand. For example, the shortest fragment is comprised of a single base, such base being the first base in the sequence. The longest fragment terminates in a base which is the last base of the sequence. The relative lengths of the framents are resolved by electrophoresis thereby enabling the sequence to be ascertained.
Further details of the Maxam/Gilbert sequencing method will be described in connection with FIGS. 1A through 1G. Single-stranded DNA is comprised of four bases, A (Adenine), G (Guanine), C (Cytosine) and T (Thymine). These bases are arranged in the strand to form a sequence. The first step of the sequencing process is to isolate a large number of strands having identical DNA sequences. By way of example, FIG. 1A schematically depicts, in very simplified terms, several DNA strands having the identical sequence CAAGAGATAC. In actual practice, a large number of strands will be isolated. Next, each strand is terminally labeled with a radioactive tracer such as P32, as shown schematically in FIG. 1B.
Once the strands have been labeled, the strands are separated into four groups. Each group is then chemically treated to cleave the base-to-base bonds in a particular way. A first group is placed in a first vial, labeled for convenience as Vial A. Vial A contains chemicals, well known in the art, which, in simplified terms, cause the strands in the vial to cleave the bond at the right of one of the A bases. Thus, the exemplary sequence shown in FIGS. 1A and 1B will produce, with equal probability, the following five different labeled fragments depicted in FIG. 1C: P32CA; P32CAA; P32CAAGA; P32CAAGAGA; and P32CAAGAGATA. Fragments without a P32 tracer are also present in the vial, but will not be detected in the electrophoresis process.
The second group of identically labeled strands is placed in a second vial, labeled Vial C, which contains chemicals, well known in the art, which cause the strands in the vial to break the sequence at the right of one of the C bases. The following two labeled fragments will be produced, as shown in FIG. 1D: P32C; and P32CAAGAGATAC.
The third and fourth groups of strands are placed in third and fourth vials, labeled Vials G and T, respectively. Vials G and T contain chemicals, which cause the strands in the vials to break the bonds to the right of G and T bases, respectively. FIG. 1F depicts the following two Vial G labeled fragments which result: P32CAAG and P32CAAGAC. FIG. 1F shows the single Vial T labeled fragment which is produced: P32 CAAGAGAT.
The tagged P32 fragments are then separated by size using conventional electrophoresis. A separate gel track, typically one meter in length is provided for each of the four groups of fragments. Each track has a square well at the top for the initial placement of the DNA fragments. A uniform voltage is applied across the length of the gel, causing the fragments to travel along the gel track with a velocity approximately according to the following equation: EQU V=(K) (V) (-log (N)+A) (1)
where K and A are positive constants, V is the applied voltage and N is the number of bases contained in the fragment.
It can be seen from equation (1) that the velocity of the fragments is a non-linear function of the applied voltage, with the smaller fragments traveling at the higher velocities.
After an elapsed time interval, the fragments will be distributed in subgroups along the length of each of the parallel gel tracks in accordance with the length of the fragments in the subgroup. The gel is then removed and exposed to photographic paper to form an autoradiograph. The paper is then developed, thereby producing a visual image which shows the relative position of the subgroups of fragments along the length of the gel.
FIG. 1G illustrates a developed exposure which was made for the FIG. 1A DNA sequence in accordance with the previously-described autoradiography procedure. The tagged fragments were initially positioned at the top of the exposure, with the Vial A track being positioned along the left edge of the exposure, followed by the Vial C, G and T tracks.
The subgroup comprised of the smallest fragments will be comprised of the single base adjacent the P32 label of the original strand as shown in FIG. 1D. This subgroup of fragments will have traveled the greatest distance, as indicated by equation (1). Since the smallest fragment came from Vial C, the first base of the sequence is a C base. The next smallest subgroup of fragments will be comprised of fragments having two bases, including first base C followed by a second base. As can be seen from FIG. 1G, the next smallest (fastest) fragment came from Vial A, therefor the second base of the sequence is an A. This process is continued for each of the remaining eight bases. The final sequence is P32CAAGAGATAC which corresponds to the FIG. 1A sequence.
It was previously assumed that vials G and T originally contained fragments which terminated in G at T bases, respectively. In actual practice and in accordance with conventional chemical processes, vial G will probably contain fragments which terminate in both G and A fragments and vial T will contain fragments which terminate in both T and C fragments. In that event, fragments terminating in T bases can be uniquely identified by observing the presence of fragments at a particular position on the exposure along the vial T track and the absence of fragments at the corresponding position along the C vial track. The position of fragments terminating in G bases can be uniquely determined in a similar manner.
Conventional gel electrophoresis utilizing autoradiography is quite time consuming and very labor intensive. The electrophoresis gel must be carefully prepared so as to provide a uniform structure through which the fragments pass. Any nonuniformity may result in sequencing errors. A gel approximately 1 meter long is capable of determining roughly 100 bases of a sequence. For a DNA sequence of 1000 bases, the procedure requires 5-10 gel runs lasting 8 to 16 hours. After each gel is run, the gel must be separated from its supporting glass plate and exposed for 8 to 48 hours with photographic paper. After the DNA sequence is obtained, it is typically manually entered into a computer for further analysis. The entire procedure, which must be performed by a skilled technician, requires at least a week of applied time and an elapsed time of several weeks. In addition, evaluation of the autoradiation photographs and entry of the sequence into a computer is susceptible to human error.
Some attempts have been made to overcome the above-noted limitation of the Maxam/Gilbert sequencing procedure. For example, it is believed the automated imaging techniques have been utilized to analyze the autoradiation photographs. Despite such advances, the principal shortcomings of the Maxam/Gilbert procedure remain.
The present apparatus and method for DNA and RNA sequencing overcomes the limitations of the Maxam/Gilbert procedure. The time required to obtain a DNA sequence is greatly reduced. Moreover, the procedure can be carried out by persons having limited training. In addition, overall accuracy is improved inasmuch as it is not necessary to interpret a photograph and manually enter data into a computer. These and other advantages of the subject invention will become apparent to those having average skill in the art upon reading the following Best Mode For Carrying Out The Invention.